The effect of glucocorticoid withdrawal after long-term treatment on POMC and its transcription factors
Abduladim Eltobgi, Alia Munir, Neil Smith, Lee Abbott & John Newell-Price
Background: Proopiomelanocortin gene (POMC) and its cleaved product adrenocorticotrophin hormone (ACTH) play an important role in regulation of the hypothalamo-pituitary-adrenal (HPA) axis. ACTH stimulates the adrenal glands to release glucocorticoids. Glucocorticoid has a suppressive effect on ACTH and POMC. This suppression remains long after withdrawal, and may lead to adrenocortical insufficiency. POMC expression is dependent on the activities of some of the positively acting transcription factors (NeuroD1, Tpit, Ptx1 and Nur77).
Hypothesis: Long-term glucocorticoids inhibit expression of POMC and its transcription factors, by altering the chromatin configuration via de-novo methylation of the POMC promoter.
Methods: AtT20 cells were cultured in standard media supplemented with dexamethasone (DEX) 1 μM and 100 nM, for intervals ranging from 72 hours to 40 weeks. After 40 weeks of treatment, the DEX was withdrawn. DNA and RNA were extracted and qPCR performed. ELISA was used to assess the ACTH levels. POMC promoter methylation was assessed by bisulphite sequencing.
Results: POMC expression is down-regulated after 72 hours of DEX treatment. NeuroD1 and Tpit expression are inhibited during 40 weeks of DEX exposure. ACTH levels were significantly suppressed. DEX did not show any effect on Ptx1 and Nur77. No methylation of the POMC promoter was detected. At 3 weeks post withdrawal, POMC, NeuroD1 and Tpit mRNA remained suppressed. The recovery of ACTH levels was not observed in these cells.
Conclusions: Glucocorticoid inhibits POMC, NeuroD1 and Tpit expression. The knock down of POMC is followed by suppression of ACTH. No recovery of POMC, Neuro D1, and Tpit mRNA or ACTH, was seen at 3 weeks post withdrawal of the 40 week treatment. DEX does not cause de-novo methylation in this region of the POMC promoter at 40 weeks. It is likely that the down regulation of NeuroD1 and Tpit are implicated and methylation of these genes may play a role.